| Wheat storage protein mainly included glutenin and gliadin , about 80% of wheat seed protein, and constituted the main ingredient of gluten. Quality of gluten was determined by quantity and scale of glutenin and gliadin. Gliadin was one of the main ingredient of gluten, about 40%~50% of total amount of protein, the quality of effects had been issue and difficulty of research.The reasons was not only to the difficulty of separation, but also with rich diversity of the genes family and reconsitution of the protein space. Though different scholars had severally conflicting conclusion, most of the research results showed that gliadin reduced stability of the dough.Specific primers were designed according to conserved ends of the coding region of knownα-gliadin genes, the genomic DNA was a template for PCR, the molecular structure of gene and its kinship were analyzed;Target fragment was PCR with primers of expression of which didn't PCR signal peptide, then recovered target fragment and was connected with express vector. The subunit ofα-gliadin was expressed by E. coli effectively and was got by purifying, then integrated into the flour by the oxidation reduction reaction, rheological properties were tested by micro 4g farinograph in order to research the effect ofα-gliadin to dough, the results were as follows:1. Seven DNA sequences were cloned from a strong gluten wheat variety Shaan 253, namedα-Agli-1~7(GenBank NO. was GQ891681~ GQ891687)respectively, except forα-Agli-3 andα-Agli-5, the remaining five genes were pseudogene of which genetic coding region had stop codons.The sequence analysis showed that the identity ofα-Agli-3 andα-Agli-5 was 97.90%, the remaining five sequences was above 85%. The analysis of the distribution region of stop codons showed that they were mainly in repeated sequence region. The sites and type were CAA/CAG→TAA and CAA/CAG/GAG→TAG,the frequency of each stop codons was TAA (66%)> TAG (34%)of which CAA mutate to TAA was the highest.2. Further analysis showed that the seven cloned sequences had typical structure ofα-gliadin genes of which included: a conservative signal peptide, N-terminal repeats, poly-glutamine I region, N-terminal non-repeat region (unique domain I), poly-glutamine II region and C-terminal non-repeat region(unique domain II). Repeated region had basic repeat unit PQPQPFP and PQQPY. The differences of poly-glutamine region were length and appearance of non-glutamine.α-Agli-4,α-Agli-6 andα-Agli-7 had six cysteine residues,α-Agli-1 andα-Agli-2 had five and nine cysteine residues respectively, may be caused by frameshift mutations;α-Agli-3 andα-Agli-5 had seven cysteine residues of which in the unique domain II cysteine was instead of serine due to C→G in the codon of the sixth amino acid, which maybe form intermolecular disulfide bond.3. Analyzedα-Agli-3 andα-Agli-5 sequence,α-Agli-5 was expressed in E.coli. Specific expressed primers were designed according to its coding sequence to amplify the target fragment.Prokaryotic expression vector pET32a-α-Agli-5 was constructed, the positive clones of recombinant plasmid pET32a-α-Agli-5 was transformed into expression host strain BL21(DE3)competent cells, after inducting by IPTG of 1mmoL, 28℃6 h culture , supernatant of the total bacterial proteins was extracted then 12% SDS-PAGE electrophoresis and western blot were used to test the product, they both appeared a 55 kD protein band of which was the purpose expression protein, and the results was in accord with the predicted molecular weight.4. A large number of expressed protein was prepared and purified by ?KTA purifier 100 protein chromatography system.Purified protein was integrated into the flour by the oxidation reduction reaction, rheological properties were tested by micro 4 g farinograph in order to research the effect ofα-gliadin to dough. The results show that flexibility of gluten increased but strength of gluten decreaded whenα-gliadinα-Agli-5 was added.
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